LTK Antibody

What is LTK and why is it important in cellular research?

LTK (Leukocyte tyrosine kinase, also known as protein tyrosine kinase 1 or TYK1) is a glycoprotein member of the tyrosine protein kinase family, belonging to the insulin receptor subfamily of proteins . It functions as an ER-resident receptor tyrosine kinase that regulates COPII-dependent trafficking, making it the first identified ER-resident receptor tyrosine kinase with this function . LTK is particularly important in research because it represents a potential druggable proteostasis regulator . It is expressed in various tissues including lymphocytes, cerebral cortex neurons, and cardiomyocytes, where it plays a role in cellular hypertrophy .

How can I detect endogenous LTK in my samples?

Endogenous LTK detection requires specific antibodies and appropriate techniques. Immunofluorescence can be performed using anti-LTK antibodies, with HepG2 cells being particularly suitable due to their high LTK expression levels and minimal ALK expression (which prevents cross-reactivity issues) . For Western blotting, available antibodies may not consistently detect endogenous LTK, so validation is essential . Alternatively, overexpression of tagged LTK constructs (e.g., flag-tagged) can facilitate detection in cellular studies . When performing immunofluorescence, co-staining with ER markers such as CLIMP63 can confirm the ER localization of LTK .

What are the common applications for LTK antibodies?

LTK antibodies are used in various research applications including:

For optimal results, antibody dilutions should be determined for each specific application and experimental system .

How do I validate the specificity of an LTK antibody?

Validating LTK antibody specificity is crucial for reliable experimental results. The most common validation approach is to perform knockdown experiments using LTK-specific siRNAs and demonstrate a reduction in signal intensity compared to control conditions . This approach has been successfully used to validate antibody specificity in immunofluorescence studies . Additionally, comparison of staining patterns with known LTK subcellular localization (primarily ER) can provide further validation . For Western blot applications, the detection of a specific band at the expected molecular weight (~70 kDa) that diminishes upon LTK knockdown confirms antibody specificity .

How can I distinguish between different LTK isoforms using antibodies?

LTK exists in multiple isoforms with molecular weights ranging between 50 and 100 kDa in SDS-PAGE . Distinguishing between these isoforms requires:

• Selection of epitope-specific antibodies: Choose antibodies targeting regions that differ between isoforms. The database indicates several isoforms including:

  • Isoforms with a common deletion of aa 274-334

  • An isoform with an alternative start site at Met406

  • An isoform with a 51 aa substitution for aa 171-864

  • A 50 kDa isoform with a 28 aa substitution for aa 449-864

• High-resolution gel electrophoresis: Use gradient gels (e.g., 4-20%) to achieve better separation of closely migrating isoforms.

• Isoform-specific knockdown: Employ isoform-specific siRNAs to validate antibody specificity for particular variants.

• Mass spectrometry validation: Following immunoprecipitation with anti-LTK antibodies, mass spectrometry can definitively identify specific isoforms present in your sample.

When using commercial antibodies, verify the immunogen sequence to determine which isoforms the antibody is likely to recognize .

What methodological considerations are important when studying LTK phosphorylation states?

Studying LTK phosphorylation requires careful experimental design:

• Antibody selection: Use phospho-specific antibodies that recognize specific phosphorylated tyrosine residues in LTK. The cytoplasmic region of LTK possesses multiple phosphotyrosines that interact with downstream signaling molecules .

• Phosphatase inhibitors: Include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) in all lysis buffers to preserve phosphorylation states.

• Stimulation protocols: LTK activation may require specific stimulation conditions. Unlike its relative ALK, LTK is not readily accessible to extracellular ligands due to its ER localization .

• Sample preparation: Rapid sample processing is essential to prevent dephosphorylation. Use hot SDS sample buffer or direct lysis in Laemmli buffer for immediate denaturation of phosphatases.

• Controls: Include samples treated with phosphatase to confirm the specificity of phospho-specific antibodies.

• Detection methods: Enhanced chemiluminescence with sensitive substrates or fluorescent secondary antibodies often provides better quantification of phosphorylation signals.

Remember that LTK's ER localization means its activation mechanisms likely differ from typical cell surface receptor tyrosine kinases .

How can I effectively study LTK's role in ER-to-Golgi trafficking using antibodies?

LTK regulates ER-to-Golgi trafficking through modulation of ER exit sites (ERESs) . To study this function:

• Combined knockdown and imaging approach:

  • Deplete LTK using validated siRNAs (such as siRNA #3 mentioned in the literature)

  • Quantify the number of ERESs using antibodies against ERES markers (e.g., Sec31)

  • Expected outcome: 30-40% reduction in ERES number in LTK-depleted cells (HepG2 and HeLa)

• Pharmacological inhibition:

  • Treat cells with LTK inhibitors such as alectinib or crizotinib (30 min treatment)

  • Analyze ERES numbers as above

  • Note: Use cell lines negative for ALK (e.g., HepG2) to ensure effects are LTK-specific

• Trafficking assays:

  • Implement cargo trafficking assays (e.g., VSVG-GFP or other reporter systems)

  • Compare trafficking kinetics between control and LTK-depleted/inhibited cells

  • Combine with immunofluorescence using anti-LTK antibodies to correlate LTK levels with trafficking efficiency

• Co-localization studies:

  • Perform dual immunofluorescence with antibodies against LTK and ERES/ERGIC/Golgi markers

  • Note that approximately 10% of cells show weak co-localization between LTK and the ERES marker Sec31

These methodologies can be combined with live-cell imaging approaches for dynamic studies of trafficking regulation by LTK.

What are the challenges and solutions for using LTK antibodies in tissue samples compared to cell lines?

Studying LTK in tissue samples presents several challenges not encountered in cell line research:

When working with neuronal tissues, specific staining of LTK is typically localized to neurons, which can serve as an internal validation of staining specificity .

How do I resolve weak or non-specific LTK antibody signals in Western blots?

When encountering weak or non-specific LTK signals, implement these methodological solutions:

• Sample preparation optimization:

  • Use RIPA or stronger lysis buffers with protease inhibitors

  • For membrane proteins like LTK, consider specialized membrane protein extraction buffers

  • Include proper controls (LTK-overexpressing cells, LTK knockdown samples)

• Antibody selection and optimization:

  • Test multiple antibodies targeting different epitopes of LTK

  • Optimize antibody concentration through titration experiments

  • For human LTK, Sheep Anti-Human LTK Antigen Affinity-purified Polyclonal Antibody has been successful in detecting a specific band at approximately 70 kDa

• Blotting conditions:

  • Perform experiments under reducing conditions

  • Use Immunoblot Buffer Group 8 for optimal results with some antibodies

  • Transfer conditions may need optimization for this transmembrane protein

• Detection enhancement:

  • Use more sensitive ECL substrates

  • Consider longer exposure times

  • Try signal enhancers like protein-free blocking buffers

• Membrane selection:

  • PVDF membranes have been successfully used for LTK detection

  • Ensure proper activation of membranes before transfer

Remember that available antibodies may not consistently detect endogenous LTK by immunoblotting, so overexpression systems may be necessary for some experiments .

What controls should be included when studying LTK's influence on the ER stress response?

When investigating LTK's role in ER stress responses, include these essential controls:

• Positive controls for ER stress induction:

  • Thapsigargin treatment (as used in previous studies)

  • Tunicamycin treatment (glycosylation inhibitor)

  • Glucose deprivation

• Genetic controls:

  • LTK knockdown cells (using validated siRNAs)

  • LTK-overexpressing cells

  • Rescue experiments with wild-type vs. kinase-dead LTK

• Pharmacological controls:

  • LTK inhibitors (alectinib, crizotinib)

  • Control compounds with similar structures but no LTK inhibitory activity

• ER stress markers to monitor:

  • XBP1 splicing (XBP1s levels have been shown to increase with LTK inhibition during thapsigargin treatment)

  • BiP/GRP78 upregulation

  • PERK phosphorylation

  • ATF6 cleavage

  • CHOP expression

• Temporal controls:

  • Monitor stress markers at multiple time points

  • Distinguish between acute and chronic effects

Previous research has indicated that LTK inhibition increases the ER stress response (measured by XBP1s levels) in cells treated with thapsigargin, suggesting LTK may help cells cope with proteostatic challenges .

How can I determine if LTK antibodies cross-react with the closely related ALK receptor?

To assess and prevent cross-reactivity between LTK and ALK antibodies:

• Cell line selection:

  • Use HepG2 cells for LTK studies as they express high levels of LTK but are essentially ALK-negative

  • Include both ALK-positive and ALK-negative cell lines as controls

• Knockdown verification:

  • Perform siRNA knockdown of LTK and test if antibody signal decreases

  • Test antibody signal in ALK knockdown cells (should remain unchanged if specific for LTK)

• Epitope analysis:

  • Select antibodies targeting regions with minimal sequence homology between LTK and ALK

  • N-terminal antibodies may provide better specificity as this region typically has less conservation

• Recombinant protein controls:

  • Test antibodies against purified recombinant LTK and ALK proteins

  • Perform competitive binding assays with these proteins

• Known differential properties:

  • LTK is completely sensitive to EndoH treatment, while ALK is only partially sensitive (~60%)

  • LTK is insensitive to PNGase F treatment of intact cells, while ALK is sensitive

  • These differential properties can be used to distinguish between the proteins

These approaches will help ensure the specificity of your antibody for LTK-focused research.

What are the optimal conditions for immunoprecipitating LTK for downstream phosphorylation analysis?

For successful LTK immunoprecipitation and phosphorylation analysis:

• Lysis buffer composition:

  • Use NP-40 or RIPA buffer with phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Include protease inhibitor cocktail

  • Buffer pH should be maintained at 7.4-7.6

• Antibody selection:

  • Choose antibodies validated for immunoprecipitation

  • Antigen affinity-purified antibodies generally work best

  • For tagged LTK, anti-tag antibodies (e.g., anti-FLAG) often provide cleaner results

• Precipitation protocol:

  • Pre-clear lysates with protein A/G beads

  • For rabbit polyclonal LTK antibodies, protein A beads are optimal

  • For sheep antibodies like AF4664, protein G columns have been effectively used

  • Incubate antibody with lysate overnight at 4°C for maximum capture

• Washing conditions:

  • Use stringent washes to reduce background

  • Include phosphatase inhibitors in all wash buffers

  • Maintain cold temperature throughout to preserve phosphorylation

• Elution methods:

  • For immunoblotting: direct elution in SDS sample buffer

  • For mass spectrometry: gentler elution using peptide competition or low pH buffers with immediate neutralization

• Detection strategies:

  • Probe blots with phospho-specific antibodies

  • Consider using anti-phosphotyrosine antibodies (4G10, pY100) to detect general tyrosine phosphorylation

  • For comprehensive phosphosite mapping, use phospho-enrichment followed by mass spectrometry

Remember that LTK undergoes dimerization and trimerization under certain circumstances, which may affect immunoprecipitation efficiency .

How can LTK antibodies be used to investigate the role of LTK in disease models?

LTK antibodies are valuable tools for investigating its role in disease models, particularly in systemic lupus erythematosus (SLE) and cancer:

• Systemic Lupus Erythematosus (SLE) research:

  • Immunohistochemistry of lymphoid tissues from SLE patients/models to quantify LTK expression levels

  • Western blot analysis comparing LTK levels in normal vs. SLE B cells and plasma cells

  • Immunoprecipitation followed by phosphorylation analysis to assess activation status

  • Research focus: Gain-of-function LTK mutations have been observed in SLE patients and mice

  • Hypothesis testing: LTK overactivation may confer selective advantages to autoimmune plasma cells by enhancing secretory capacity

• Cancer research applications:

  • Tissue microarray screening with anti-LTK antibodies to identify cancer types with altered LTK expression

  • Correlation of LTK levels with cancer progression and patient outcomes

  • Dual staining with ER stress markers to evaluate proteostatic stress

  • Investigation rationale: Cancer cells are considered "addicted" to secretion due to high proteostatic challenges, making LTK a potential therapeutic target

  • Drug response monitoring: Use LTK antibodies to assess pathway inhibition after treatment with LTK inhibitors (alectinib, crizotinib)

• Methodological approaches for both disease models:

  • Knockdown/overexpression studies in relevant cell types

  • Pharmacological inhibition followed by phenotypic analysis

  • Phospho-specific antibody staining to assess activation status

  • Co-immunoprecipitation to identify disease-specific binding partners

The localization of LTK to the ER provides a unique opportunity to target ER-based signaling pathways in these disease contexts .

What methodological adaptations are needed when using LTK antibodies for high-content screening assays?

Adapting LTK antibody-based assays for high-content screening requires several methodological considerations:

• Assay optimization:

  • Determine optimal antibody concentration through titration experiments

  • Establish signal-to-background ratios for reliable quantification

  • For brain tissue screening, use the established protocol of 10 μg/mL antibody concentration with overnight incubation at 4°C

• Signal detection strategies:

  • Fluorescent secondary antibodies typically provide better quantification than chromogenic methods

  • Multi-channel imaging allows co-staining with organelle markers (e.g., ER, Golgi)

  • For tissue sections, DAB staining with hematoxylin counterstaining has been validated

• Automation adaptations:

  • Implement automated liquid handling for consistent antibody application

  • Develop computational image analysis pipelines for:

    • Quantifying LTK expression levels

    • Assessing subcellular localization

    • Measuring co-localization with markers of interest

• Controls and normalization:

  • Include on-plate controls (positive, negative, gradient standards)

  • Use nuclear or cytoplasmic markers for cell segmentation and normalization

  • Consider internal standards for plate-to-plate comparison

• Validation of hits:

  • Confirm screening results with orthogonal methods (Western blot, qPCR)

  • Use siRNA knockdown controls to validate antibody specificity in the screening format

  • For LTK inhibitor screens, include known inhibitors (alectinib, crizotinib) as benchmarks

• Endpoint selection:

  • For trafficking studies: quantify ERES number/morphology (expected 30-40% reduction upon LTK inhibition)

  • For ER stress response: measure XBP1s levels, which increase with LTK inhibition during stress

  • For protein secretion assays: monitor reporter protein trafficking from ER to Golgi

These methodological adaptations will enable reliable high-throughput screening using LTK antibodies in various experimental contexts.

How can novel proximity labeling approaches be combined with LTK antibodies to map the LTK interactome?

Combining proximity labeling with LTK antibodies offers powerful approaches to map the LTK interactome:

• BioID/TurboID methodology:

  • Generate LTK-BioID fusion constructs (ensuring ER localization is maintained)

  • Express in relevant cell types (e.g., HepG2, lymphocytes, neurons)

  • Induce biotinylation of proximal proteins

  • Purify biotinylated proteins using streptavidin

  • Verify results with LTK antibodies in co-immunoprecipitation experiments

  • Focus on ER-localization: Since LTK is ER-resident, proximity labeling will help identify ER-specific interactors

• APEX2-based approaches:

  • Create LTK-APEX2 fusions

  • Trigger brief biotinylation (1 minute) to capture transient interactions

  • Particularly useful for mapping dynamic interactions during ER stress

  • Validate findings with traditional co-immunoprecipitation using LTK antibodies

• Split-BioID systems:

  • Design split constructs to detect specific protein-protein interactions

  • Especially valuable for studying LTK dimerization/trimerization, which has been reported under certain conditions

  • Map interaction interface regions

• Validation and analysis strategies:

  • Confirm proximity labeling findings with traditional antibody-based co-immunoprecipitation

  • Use ERES-specific markers (e.g., Sec31) to validate the 10% of cells showing LTK-ERES co-localization

  • Employ knockdown studies of identified interactors to assess functional relevance to COPII-dependent trafficking

• Comparative interactomics:

  • Compare LTK interactome under normal conditions versus:

    • ER stress conditions (thapsigargin treatment)

    • LTK inhibitor treatment (alectinib, crizotinib)

    • Disease models (SLE, cancer cell lines)

This integrated approach will provide unprecedented insights into LTK's protein interaction network within the ER environment.

What are the latest approaches for studying the functional impact of LTK post-translational modifications using specific antibodies?

Advanced approaches for studying LTK post-translational modifications include:

• Phospho-specific antibody development and application:

  • Generate antibodies against specific phosphorylated tyrosine residues in LTK

  • Map the temporal sequence of tyrosine phosphorylation events

  • Correlate specific phosphorylation sites with downstream pathway activation

  • Focus on the multiple phosphotyrosines in the cytoplasmic region (aa 450-864) that interact with downstream signaling molecules

• Mass spectrometry-guided antibody approaches:

  • Use phosphoproteomics to identify novel PTM sites on LTK

  • Develop and validate site-specific antibodies

  • Apply these antibodies to:

    • Track modification dynamics during ER stress

    • Monitor changes during inhibitor treatment

    • Compare normal vs. disease states

• Targeted protein engineering combined with antibody detection:

  • Generate LTK mutants lacking specific modification sites

  • Employ site-specific antibodies to confirm loss of modification

  • Assess functional consequences on ERES formation (30-40% reduction is expected with LTK depletion)

  • Determine effects on ER stress responses (XBP1s levels)

• Proximity ligation assays (PLA):

  • Combine antibodies against LTK and potential binding partners

  • Use PLA to visualize and quantify specific interactions

  • Apply in situ to tissues and cells to maintain native context

  • Particularly valuable for studying how PTMs alter protein interactions

• Integrative approaches:

  • Correlate PTM patterns with:

    • LTK enzymatic activity

    • Binding partner preferences

    • Subcellular localization

    • Degradation kinetics

These methodologies provide a comprehensive toolkit for understanding how post-translational modifications regulate LTK function in normal physiology and disease states.

How might single-cell analysis using LTK antibodies reveal heterogeneity in LTK expression and function?

Single-cell analysis with LTK antibodies can reveal important functional heterogeneity:

• Single-cell immunofluorescence microscopy:

  • Quantify LTK expression levels in individual cells

  • Examine subcellular distribution patterns

  • Correlate with ER morphology and ERES formation

  • Investigate the observation that approximately 10% of cells show weak co-localization between LTK and the ERES marker Sec31

  • Determine if this represents a distinct functional subpopulation

• Flow cytometry and cell sorting:

  • Optimize intracellular staining protocols for LTK detection

  • Sort cells based on LTK expression levels

  • Characterize sorted populations for:

    • Secretory capacity

    • ER stress responses

    • Sensitivity to LTK inhibitors

• Single-cell Western blotting:

  • Adapt LTK antibody protocols for microfluidic Western platforms

  • Compare LTK expression and phosphorylation states at single-cell resolution

  • Correlate with cellular phenotypes

• Imaging mass cytometry:

  • Label tissue sections with metal-tagged LTK antibodies

  • Simultaneously detect multiple markers at subcellular resolution

  • Map LTK expression in relation to tissue microenvironment

  • Particularly valuable for heterogeneous tissues like brain, where LTK is localized to neurons

• Correlation with single-cell transcriptomics:

  • Integrate antibody-based protein detection with scRNA-seq data

  • Identify transcriptional signatures associated with varying LTK expression

  • Investigate post-transcriptional regulation by comparing mRNA and protein levels

This multi-modal single-cell approach will provide unprecedented insights into the functional significance of cell-to-cell variability in LTK expression, potentially revealing specialized subpopulations with distinct roles in secretion regulation and ER homeostasis.

How can LTK antibodies contribute to developing novel therapeutic approaches targeting ER proteostasis?

LTK antibodies can facilitate the development of ER proteostasis-targeted therapeutics through:

• Target validation strategies:

  • Use antibodies to correlate LTK expression/activation with disease severity

  • Perform IHC studies across tissue microarrays of various diseases with ER stress components

  • Validate LTK as a drug target based on its role as the first identified ER-resident receptor tyrosine kinase regulating COPII-dependent trafficking

• Therapeutic antibody development:

  • Although LTK is primarily ER-localized, investigate potential exofacial epitopes for therapeutic antibody targeting

  • Explore antibody-drug conjugates targeted to cells overexpressing LTK

  • Develop cell-penetrating antibodies or antibody fragments to reach intracellular LTK

• Small molecule drug discovery support:

  • Implement antibody-based assays to screen for compounds that modulate LTK activity

  • Use phospho-specific antibodies to monitor target engagement

  • Develop competitive binding assays to identify compounds that disrupt specific protein-protein interactions

  • Build on existing knowledge of LTK inhibitors (alectinib, crizotinib) that reduce ERES numbers

• Disease-specific applications:

  • Systemic lupus erythematosus: Investigate how gain-of-function LTK mutations affect ER proteostasis in plasma cells

  • Cancer: Explore LTK inhibition as a strategy to induce ER stress in cancer cells already challenged by proteostatic stress

  • Neurodegenerative diseases: Study LTK's role in neuronal ER homeostasis, given its expression in cerebral cortex neurons

• Combination therapy development:

  • Use antibody-based assays to identify synergistic targets that enhance LTK inhibition effects

  • Test combinations of LTK inhibitors with other drugs targeting ER stress response pathways

  • Monitor XBP1s levels as a readout, which increase with LTK inhibition during thapsigargin treatment

These approaches position LTK antibodies as crucial tools in developing a new class of therapeutics targeting ER proteostasis regulation.

What methodological innovations might improve the study of LTK's dynamic regulation during ER stress responses?

Advanced methodologies for studying LTK dynamics during ER stress include:

• Biosensor development:

  • Design FRET-based biosensors to monitor LTK conformational changes

  • Develop activity reporters based on known LTK substrates

  • Create tension sensors to detect mechanical forces during ER membrane remodeling

  • Validate these tools using existing antibodies as references

• Live-cell super-resolution microscopy:

  • Implement antibody fragment-based labeling for live-cell imaging

  • Track LTK dynamics relative to ERES formation and dissolution

  • Correlate with ER stress markers in real-time

  • Investigate the dynamic nature of the weak co-localization observed between LTK and Sec31 in 10% of cells

• Conditional protein regulation:

  • Develop rapid, reversible LTK inhibition systems (e.g., chemical genetic approaches)

  • Create optogenetic LTK activation/inhibition tools

  • Monitor acute effects on ERES numbers (expected 30-40% reduction upon inhibition)

  • Track consequences for XBP1 splicing and other ER stress markers

• Microfluidic approaches:

  • Design chambers for controlled application/removal of ER stressors

  • Combine with live-cell antibody-based detection methods

  • Monitor LTK expression, localization, and activation in real-time during stress induction and recovery

• Spatial transcriptomics integration:

  • Correlate LTK protein levels (detected by antibodies) with local transcriptional responses

  • Map spatial relationships between LTK activity and ER stress response gene expression

  • Particularly relevant in tissues with heterogeneous LTK expression like brain

These methodological innovations will provide unprecedented insights into the temporal dynamics of LTK function during ER stress, potentially revealing new therapeutic windows for intervention in diseases characterized by ER dysfunction.

How might comparative studies using LTK antibodies across species contribute to our understanding of evolutionary conservation in ER-based signaling?

Cross-species LTK antibody studies can reveal evolutionary aspects of ER signaling:

• Epitope conservation analysis:

  • Test antibody cross-reactivity with LTK orthologs from different species

  • Map conserved functional domains using epitope-specific antibodies

  • Compare with sequence analysis revealing that human LTK shares 75% amino acid identity with mouse LTK over the region aa 17-424

• Functional conservation studies:

  • Compare LTK subcellular localization across species using validated antibodies

  • Determine if ER localization is evolutionarily conserved

  • Assess ERES regulation function in diverse species

  • Test whether inhibitors produce similar effects (30-40% ERES reduction) across species

• Tissue expression pattern comparison:

  • Use antibodies to map LTK expression across tissues in different species

  • Compare with human expression in lymphocytes, cerebral cortex neurons, and cardiomyocytes

  • Identify species-specific expression patterns that might indicate specialized functions

• Disease model translation:

  • Test antibodies in various species models of SLE and cancer

  • Determine if the proposed role of LTK in helping cells cope with secretory load is conserved

  • Evaluate cross-species validity of LTK as a therapeutic target

• Developmental regulation:

  • Map LTK expression during embryonic development across species

  • Identify conserved vs. divergent temporal expression patterns

  • Link to the development of secretory tissues

This evolutionary perspective will provide valuable insights into the fundamental importance of LTK-mediated ER signaling and help distinguish conserved core functions from species-specific adaptations.

What are the relative advantages and limitations of different imaging techniques when using LTK antibodies?

Understanding these comparative strengths and limitations enables selection of the optimal imaging approach for specific LTK research questions.

How do different antibody-based proteomic approaches compare for studying LTK-dependent pathways?

Each approach offers distinct advantages for investigating different aspects of LTK biology, from protein interactions to signaling pathway regulation.